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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2016/11/30 10:16:03」(JST)

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Functional domains in the Klenow Fragment (left) and DNA Polymerase I (PDB).

The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. First reported in 1970,^[1] it retains the 5' → 3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity.

The other smaller fragment formed when DNA polymerase I from E. coli is cleaved by subtilisin retains the 5' → 3' exonuclease activity but does not have the other two activities exhibited by the Klenow fragment (i.e. 5' → 3' polymerase activity, and 3' → 5' exonuclease activity).

Research

Because the 5' → 3' exonuclease activity of DNA polymerase I from E. coli makes it unsuitable for many applications, the Klenow fragment, which lacks this activity, can be very useful in research. The Klenow fragment is extremely useful for research-based tasks such as:

Synthesis of double-stranded DNA from single-stranded templates
Filling in receded 3' ends of DNA fragments to make 5' overhang blunt
Digesting away protruding 3' overhangs
Preparation of radioactive DNA probes

The Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in the polymerase chain reaction (PCR) process,^[2] before being replaced by thermostable enzymes such as Taq polymerase.^[3]

The exo- Klenow fragment

Just as the 5' → 3' exonuclease activity of DNA polymerase I from E.coli can be undesirable, the 3' → 5' exonuclease activity of Klenow fragment can also be undesirable for certain applications. This problem can be overcome by introducing mutations in the gene that encodes Klenow. This results in forms of the enzyme being expressed that retain 5' → 3' polymerase activity, but lack any exonuclease activity (5' → 3' or 3' → 5'). This form of the enzyme is called the exo- Klenow fragment.

The exo-Klenow fragment is used in some fluorescent labeling reactions for microarray.

References

^ Klenow H and Henningsen I (1970). "Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis". Proc. Natl. Acad. Sci. USA. 65 (1): 168–175. doi:10.1073/pnas.65.1.168. PMC 286206. PMID 4905667.
^ Saiki RK, et al. (1985). "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia". Science. 230: 1350–54. doi:10.1126/science.2999980. PMID 2999980.
^ Saiki; et al. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239: 487–91. doi:10.1126/science.2448875. PMID 2448875.

External links

Klenow Fragment at the US National Library of Medicine Medical Subject Headings (MeSH)
Diagram at vivo.colostate.edu

English Journal

Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy.

Chao J1, Zhang P, Wang Q, Wu N, Zhang F, Hu J, Fan CH, Li B.
Nanoscale.Nanoscale.2016 Mar 21;8(11):5842-6. doi: 10.1039/c5nr06544e. Epub 2016 Mar 2.
We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon complet
PMID 26932823

Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

Suzuki M1, Kino K2, Kawada T1, Oyoshi T3, Morikawa M1, Kobayashi T1, Miyazawa H1.
Journal of biochemistry.J Biochem.2016 Mar;159(3):323-9. doi: 10.1093/jb/mvv103. Epub 2015 Oct 21.
Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized
PMID 26491064

Synthesis of Pyridone-based Nucleoside Analogues as Substrates or Inhibitors of DNA Polymerases.

Tauraitė D1, Ražanas R1, Mikalkėnas A2, Serva S1,2, Meškys R1.
Nucleosides, nucleotides & nucleic acids.Nucleosides Nucleotides Nucleic Acids.2016 Feb 8:1-15. [Epub ahead of print]
The synthesis and characterization of novel acyclic and cyclic pyridone-based nucleosides and nucleotides is described. In total, seven nucleosides and four nucleotides were synthesized. None of the tested nucleosides showed inhibitory properties against Klenow exo- polymerase and M.MuLV and HIV-1 r
PMID 26854871

Japanese Journal

Simple, Colorimetric Detection of MicroRNA Based on Target Amplification and DNAzyme

YAN Chunyan,JIANG Cheng,JIANG Jianhui [他],YU Ruqin
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 29(6), 605-610, 2013-06-10
… In the presence of the target, the HP was opened, and then the isothermal circular strand-displacement process occurred with the help of a primer, deoxynucleotide solution mixture (dNTPs), Klenow fragment exo− polymerase, and Nb.BbvCI nicking enzyme. …
NAID 10031177782

Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo- ) DNA polymerase

Nucleic Acids Research 37(17), 5602-5609, 2009-07
… In our previous communication we reported the enzymatic recognition of unnatural imidazopyridopyrimidine:naphthyridine (Im:Na) base pairs, i.e. ImON:NaNO and ImNO:NaON, using the Klenow fragment exo [KF (exo )]. …
NAID 80020653798

Requirements for frameshift (deletion) during translesion synthesis (第32回〔日本環境変異原学会〕大会シンポジウム(2)DNA損傷と変異のメカニズム)

Shibutani Shinya
環境変異原研究 26(2), 135-141, 2004-09-30
… The number of deletions during DNA synthesis catalyzed by the 3'→5' exonuclease-free (exo^-) Klenow fragment of Escherichia coli DNA polymerase I (pol I) was determined systematically on dG-acetylaminofluorene (dG-AAF) -modified oligodeoxynucleotides templates with different bases 3' and/or 5' to the lesion. … The formation of deletions was reduced when exo^+ Klenow fragment was used, suggesting that the proofreading function of the enzyme minimizes the deletion formation. …
NAID 110001708216